Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.     

Exploring the molecular basis of human manganese superoxide dismutase inactivation mediated by tyrosine 34 nitration

Manganese Superoxide Dismutase (MnSOD) is an essential mitochondrial antioxidant enzyme that protects organisms against oxidative damage, dismutating superoxide radical () into H₂O₂ and O₂. The active site of the protein presents a Mn ion in a distorted trigonal–bipyramidal environment, coordinated by H26, H74, H163, D159 and one ⁻OH ion or H₂O molecule. The catalytic cycle of the enzyme is a “ping-pong” mechanism involving Mn³⁺/Mn²⁺. It is known that nitration of Y34 is responsible for enzyme inactivation, and that this protein oxidative modification is found in tissues undergoing inflammatory and degenerative processes. However, the molecular basis about MnSOD tyrosine nitration affects the protein catalytic function is mostly unknown.In this work we strongly suggest, using computer simulation tools, that Y34 nitration affects protein function by restricting ligand access to the active site. In particular, deprotonation of 3-nitrotyrosine increases drastically the energetic barrier for ligand entry due to the absence of the proton.Our results for the WT and selected mutant proteins confirm that the phenolic moiety of Y34 plays a key role in assisting superoxide migration.     

Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.     

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell–cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3^−/−), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3^−/− mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3^−/− mice, but not EphB3^−/− mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.     

Towards reducing impact-induced brain injury: lessons from a computational study of army and football helmet pads

We use computational simulations to compare the impact response of different football and U.S. Army helmet pad materials. We conduct experiments to characterise the material response of different helmet pads. We simulate experimental helmet impact tests performed by the U.S. Army to validate our methods. We then simulate a cylindrical impactor striking different pads. The acceleration history of the impactor is used to calculate the head injury criterion for each pad. We conduct sensitivity studies exploring the effects of pad composition, geometry and material stiffness. We find that (1) the football pad materials do not outperform the currently used military pad material in militarily relevant impact scenarios; (2) optimal material properties for a pad depend on impact energy and (3) thicker pads perform better at all velocities. Although we considered only the isolated response of pad materials, not entire helmet systems, our analysis suggests that by using larger helmet shells with correspondingly thicker pads, impact-induced traumatic brain injury may be reduced.     

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.     

Adriamycin-mediated nitration of manganese superoxide dismutase in the central nervous system: insight into the mechanism of chemobrain

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

A modified human head model for the study of impact head injury

A recently published finite element (FE) head model is modified to consider the viscoelasticity of the meninges, the spongy and compact bone in the skull. The cerebrospinal fluid (CSF) is simulated explicitly as a hydrostatic fluid by using a surface-based fluid modelling method, which allows fluid and structure interaction. It is found that the modified model yields smoother pressure responses in a head impact simulation. The baseline model underestimated the peak von Mises stress in the brain by 15% and the peak principal stress in the skull by 33%. The increase in the maximum principal stress in the skull is mainly caused by the updation of the material's viscoelasticity, and the change in the maximum von Mises stress in the brain is mainly caused by the improvement of the CSF simulation. The study shows that the viscoelasticity of the head tissue should be considered, and that the CSF should be modelled as a fluid, when using FE analysis to study head injury due to impact.     

Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats

We investigated mechanisms by which a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 improves neurological recovery after spinal cord injury (SCI) in the rat. The effects of an anti-CD11d mAb treatment were assessed on ED-1 expression (estimating macrophage infiltration), myeloperoxidase activity (MPO, approximating neutrophil infiltration), lipid peroxidation, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicating protein nitration) expression in the spinal cord lesion after severe clip-compression injury. Protein expression was evaluated by western blotting and immunocytochemistry. Lipid peroxidation was assessed by thiobarbituric acid reactive substances (TBARS) production. After anti-CD11d mAb treatment, decreased ED-1 expression at 6-72 h after SCI indicated reduced macrophage infiltration. MPO activity (units/g tissue) was reduced significantly from 114 +/- 11 to 75 +/- 8 (- 34%) at 6 h and from 38 +/- 2 to 22 +/- 4 (- 42%) at 72 h. After SCI, anti-CD11d mAb treatment significantly reduced TBARS from 501 +/- 61 to 296 +/- 17 nm (- 41%) at 6 h and to approximately uninjured values (87 nm) at 72 h. The mAb treatment also attenuated the expression of iNOS and formation of nitrotyrosine at 6-72 h after SCI. These data indicate that anti-CD11d mAb treatment blocks intraspinal neutrophil and macrophage infiltration, reducing the intraspinal concentrations of reactive oxygen and nitrogen species. These effects likely underlie improved tissue preservation and neurological function resulting from the mAb treatment.     

Alterations in inducible 72-kDa heat shock protein and the chaperone cofactor BAG-1 in human brain after head injury

The stress response in injured brain is well characterized after experimental ischemic and traumatic brain injury (TBI); however, the induction and regulation of the stress response in humans after TBI remains largely undefined. Accordingly, we examined injured brain tissue from adult patients (n = 8) that underwent emergent surgical decompression after TBI, for alterations in the inducible 72-kDa heat shock protein (Hsp70), the constitutive 73-kDa heat shock protein (Hsc70), and isoforms of the chaperone cofactor BAG-1. Control samples (n = 6) were obtained postmortem from patients dying of causes unrelated to CNS trauma. Western blot analysis showed that Hsp70, but not Hsc70, was increased in patients after TBI versus controls. Both Hsp70 and Hsc70 coimmunoprecipitated with the cofactor BAG-1. The 33 and 46, but not the 50-kDa BAG-1 isoforms were increased in patients after TBI versus controls. The ratio of the 46/33-kDa isoforms was increased in TBI versus controls, suggesting negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain. These data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl-2 family member BAG-1, after TBI in humans.     

Neuronal uptake of nanoformulated superoxide dismutase and attenuation of angiotensin II-dependent hypertension after central administration

Excessive production of superoxide (O₂⁻) in the central nervous system has been widely implicated in the pathogenesis of cardiovascular diseases, including chronic heart failure and hypertension. In an attempt to overcome the failed therapeutic impact of currently available antioxidants in cardiovascular disease, we developed a nanomedicine-based delivery system for the O₂⁻-scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD), in which CuZnSOD protein is electrostatically bound to a poly-l-lysine (PLL₅₀)–polyethylene glycol (PEG) block copolymer to form a CuZnSOD nanozyme. Various formulations of CuZnSOD nanozyme are covalently stabilized by either reducible or nonreducible crosslinked bonds between the PLL₅₀–PEG polymers. Herein, we tested the hypothesis that PLL₅₀–PEG CuZnSOD nanozyme delivers active CuZnSOD protein to neurons and decreases blood pressure in a mouse model of angiotensin II (AngII)-dependent hypertension. As determined by electron paramagnetic resonance spectroscopy, nanozymes retain full SOD enzymatic activity compared to native CuZnSOD protein. Nonreducible CuZnSOD nanozyme delivers active CuZnSOD protein to central neurons in culture (CATH.a neurons) without inducing significant neuronal toxicity. Furthermore, in vivo studies conducted in adult male C57BL/6 mice demonstrate that hypertension established by chronic subcutaneous infusion of AngII is significantly attenuated for up to 7 days after a single intracerebroventricular injection of nonreducible nanozyme. These data indicate the efficacy of nonreducible PLL₅₀–PEG CuZnSOD nanozyme in counteracting excessive O₂⁻ and decreasing blood pressure in AngII-dependent hypertensive mice after central administration. Additionally, this study supports the further development of PLL₅₀–PEG CuZnSOD nanozyme as an antioxidant-based therapeutic option for hypertension.     

Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury

Traumatic brain injury is a common event associated with neurological dysfunction. Oxidative damage, may contribute to some of these pathologic changes. We used a specific and sensitive marker of lipid peroxidation, the isoprostane 8,12-iso-iPF(2alpha) -VI, to investigate whether local and also systemic lipid peroxidation were induced following lateral fluid percussion (FP) brain injury in the rat. Animals were anesthetized and subjected to lateral FP brain injury of moderate severity, or to sham injury as controls. Urine was collected before anesthesia (baseline), 6 and 24 h after injury. Blood was collected at baseline, 1, 6 and 24 h after injury. Animals were killed 24 h after surgery and their brains removed for biochemical analysis. No significant difference was observed at baseline (preinjury) for urine and plasma 8,12-iso-iPF(2alpha) -VI levels between injured and sham-operated animals. By contrast, plasma and urinary levels increased significantly already at 1 and further increased 24 h following brain injury, when compared to sham-operated animals. Finally, compared with sham, injured animals had a significant increase in brain 8,12-iso-iPF(2alpha) -VI levels. These results demonstrate that moderate brain injury induces widespread brain lipid peroxidation, which is accompanied by a similar increase in urine and plasma. Peripheral measurement of 8,12-iso-iPF(2alpha) -VI levels after brain injury may be a reliable marker of brain oxidative damage.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.     

颅脑创伤后大鼠脑组织脑红蛋白表达变化及其与神经元凋亡的关系研究

目的：研究大鼠弥漫性颅脑创伤后脑组织中脑红蛋白的表达变化情况,探究创伤后脑红蛋白表达变化及其与神经元凋亡的关系。方法：采用雄性SD大鼠50只,随机分为10组（n=5）空白对照组、伤后30min、1h、2h、6h、12h、24h、48h、72h和5d组。以Marmarou’s自由落体打击装置复制颅脑创伤模型,采用免疫组化技术检测伤后不同时间脑组织中脑红蛋白的表达情况及神经元凋亡相关基因Bax、Bcl-2表达情况,并对所得数据进行统计学分析。结果：致伤区皮层神经元脑红蛋白表达分别于伤后2h、72h呈现出两次高峰表达;伤后30min～1h、48～72h期间大脑皮层区脑红蛋白表达的上调均伴随着Bax/Bcl-2比值上升趋势减缓甚至呈现下降趋势。结论：弥漫性颅脑创伤后脑组织中脑红蛋白的高表达在一定程度上可以拮抗创伤应激及伤后继发缺血、缺氧性损伤所导致的神经元凋亡,在颅脑创伤的超早期（〈3h）、急性期（〈72h）可能具有一定的神经保护作用。